title: Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders.
abstract: We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved. β-Catenin gene expression and nuclear levels were analyzed by real time PCR and confocal immunofluorescence. Increased nuclear β-catenin was found in osteoblasts isolated from patients with osteoarthritis (99 ± 4 units vs. 76 ± 12, p=0.01, n=10), without differences in gene transcription, which is consistent with a post-translational down-regulation of β-catenin and decreased Wnt pathway activity. Twenty four single nucleotide polymorphisms (SNPs) of genes showing differential expression between fractures and osteoarthritis (WNT4, WNT10A, WNT16 and SFRP1) were analyzed in DNA isolated from blood of 853 patients. The genotypic frequencies were similar in both groups of patients, with no significant differences. Methylation of Wnt pathway genes was analyzed in bone tissue samples (15 with fractures and 15 with osteoarthritis) by interrogating a CpG-based methylation array. Six genes showed significant methylation differences between both groups of patients: FZD10, TBL1X, CSNK1E, WNT8A, CSNK1A1L and SFRP4. The DNA demethylating agent 5-deoxycytidine up-regulated 8 genes, including FZD10, in an osteoblast-like cell line, whereas it down-regulated other 16 genes. In conclusion, Wnt activity is reduced in patients with hip fractures, in comparison with those with osteoarthritis. It does not appear to be related to differences in the allele frequencies of the Wnt genes studied. On the other hand, methylation differences between both groups could contribute to explain the differences in Wnt activity.
authors.affiliation: Department of Internal Medicine, Hospital U.M. Valdecilla-IFIMAV, University of Cantabria, RETICEF, Santander, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: García-Ibarbia
authors.forename: Jesús
authors.initials: J
authors.lastname: Delgado-Calle
authors.forename: Iñigo
authors.initials: I
authors.lastname: Casafont
authors.forename: Javier
authors.initials: J
authors.lastname: Velasco
authors.forename: Jana
authors.initials: J
authors.lastname: Arozamena
authors.forename: María I
authors.initials: MI
authors.lastname: Pérez-Núñez
authors.forename: María A
authors.initials: MA
authors.lastname: Alonso
authors.forename: María T
authors.initials: MT
authors.lastname: Berciano
authors.forename: Fernando
authors.initials: F
authors.lastname: Ortiz
authors.forename: José L
authors.initials: JL
authors.lastname: Pérez-Castrillón
authors.forename: Agustín F
authors.initials: AF
authors.lastname: Fernández
authors.forename: Mario F
authors.initials: MF
authors.lastname: Fraga
authors.forename: María T
authors.initials: MT
authors.lastname: Zarrabeitia
authors.forename: José A
authors.initials: JA
authors.lastname: Riancho
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015870:Gene Expression; D005787:Gene Frequency; D006620:Hip Fractures / Q000209:etiology / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D015207:Osteoarthritis, Hip / Q000235:genetics* / Q000378:metabolism; D010024:Osteoporosis / Q000150:complications / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D060449:Wnt Signaling Pathway / Q000235:genetics*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D051176:beta Catenin
keywords: 5-aza-2-deoxy-azacytidine; AzadC; Bone diseases; C-terminal binding protein 1; CACYBP; CAMK2G; CSNK1A1; CSNK1A1L; CSNK1E; CTBP1; Ct; DNA methylation; FDR; FOS-like antigen 1; FOSL1; FRZB; FZD10; Fractures; GSK3B; GWAS; HWE; Hardy–Weinberg equilibrium; LRP5; PLCB3; PPP2R1A; RHOA; SFRP1; SFRP4; TATA box binding protein; TBL1X; TBP; WNT10A; WNT16; WNT4; WNT8A; Wnt; calcium/calmodulin-dependent protein kinase II gamma; calcyclin binding protein; casein kinase 1, alpha 1; casein kinase 1, alpha 1-like; casein kinase 1, epsilon; false discovery rate; frizzled homolog 10; frizzled-related protein; genome-wide association study; glycogen synthase kinase 3 beta; lipoprotein receptor related protein 5; phospholipase C, beta 3 (phosphatidylinositol-specific); protein phosphatase 2 (formerly 2A), regulatory subunit A, alpha isoform; ras homolog gene family, member A; secreted frizzled-related protein 1; secreted frizzled-related protein 4; threshold cycle; transducin (beta)-like 1X-linked; wingless-type MMTV integration site family, member 10A; wingless-type MMTV integration site family, member 16; wingless-type MMTV integration site family, member 4; wingless-type MMTV integration site family, member 8A; β-Catenin